Methods and apparatus for real-time detection and clearing of a clog

ABSTRACT

A flow cytometer apparatus and methods for detecting and clearing a clog therein are disclosed. An example method for detecting a clog may include (i) detecting, via a fault detection system of a flow cytometer, a first plurality of events associated with a first aliquot from a first sample well, (ii) determining a count of the first plurality of events associated with the first aliquot, (iii) determining whether the count of the first plurality of events is below a minimum count tolerance and (iv) (a) if the count of the first plurality of events is below the minimum count tolerance, then determining that the flow cytometer has a clog, (b) if the count of the first plurality of events is equal to or above the minimum count tolerance, then detecting a second plurality of events associated with a second aliquot from a second sample well.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/411,366 filed Jan. 20, 2017, which is a divisional application ofU.S. patent application Ser. No. 14/328,755 filed Jul. 11, 2014, thecontents of both of which are incorporated by reference herein in theirentirety.

BACKGROUND

Unless otherwise indicated herein, the materials described in thissection are not prior art to the claims in this application and are notadmitted to be prior art by inclusion in this section.

Flow cytometry is a technology employed in cell counting, cell sorting,biomarker detection and protein engineering, for example, conducted bysuspending cells in a stream of fluid and passing them by an electronicdetection apparatus. Flow cytometry allows simultaneous multiparametricanalysis of the physical and/or chemical characteristics of up tothousands of particles per second. A flow cytometer may be capable ofactively separating and isolating particles that have properties ofinterest and may permit automated quantification of set parameters.

SUMMARY

Example embodiments provide flow cytometer apparatus and methods fordetecting and/or clearing of a clog in the flow cytometer apparatus. Theapparatus and methods may beneficially permit detection of clog via afault detection system that allows data collection to pause and thenresume upon the clearing of the clog. The method for clearing the clogadvantageously permits clog removal without the need to identify theclog's location in the flow cytometer apparatus such that the flowcytometer apparatus need not be taken apart. In addition, the clogdetection and remediation methods may be automated and any clog may becleared without the need for operator intervention.

Thus, in one aspect, a flow cytometer apparatus is provided, including(i) a flow cell having a first end and a second end, (ii) a samplefluidic pathway having a first end and a second end, where the secondend of the sample fluidic pathway is coupled to the first end of theflow cell, (iv) a sample probe coupled to the first end of the samplefluidic pathway, (v) a sample pump in fluid communication with thesample fluidic pathway, (vi) a waste line having a first end and asecond end, where the first end of the waste line is coupled to the flowcell and (vii) a waste pump in fluid communication with the waste.

In a second aspect, a method for detecting a clog in a flow cytometerapparatus is provided including the steps of (i) detecting, via a faultdetection system of a flow cytometer, a first plurality of eventsassociated with a first aliquot from a first sample well, (ii)determining, via the fault detection system, a count of the firstplurality of events associated with the first aliquot, (iii)determining, via the fault detection system, whether the count of thefirst plurality of events is below a minimum count tolerance and (iv)(a) if the count of the first plurality of events is below the minimumcount tolerance, then determining, via the fault detection system, thatthe flow cytometer has a clog, (b) if the count of the first pluralityof events is equal to or above the minimum count tolerance, thendetecting, via the fault detection system, a second plurality of eventsassociated with a second aliquot from a second sample well.

In a third aspect, a method for clearing a clog from a flow cytometerapparatus is provided including the steps of (i) providing a flowcytometer system comprising (a) a flow cell having a first end and asecond end, (b) a sample fluidic pathway having a first end and a secondend, where the second end of the sample fluidic pathway is coupled tothe first end of the flow cell, (c) a sample probe coupled to the firstend of the sample fluidic pathway, (d) a sample pump in fluidcommunication with the sample fluidic pathway, (e) a waste line having afirst end and a second end, where the first end of the waste line iscoupled to the flow cell and (f) a waste pump in fluid communicationwith the waste line, (ii) activating the waste pump to apply negativepressure to one or more of the waste line, the flow cell, the sheathfluidic pathway and the sample fluidic pathway, (iii) activating thesample pump and (iv) cycling the sample probe into and out of adecontamination solution reservoir and driving a decontamination fluid,via the sample pump, through one or more of the flow cell, the samplefluidic pathway and the waste line, thereby clearing a clog.

These as well as other aspects, advantages, and alternatives, willbecome apparent to those of ordinary skill in the art by reading thefollowing detailed description, with reference where appropriate to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a flow cytometer apparatus and series ofwashing reservoirs, according to one example embodiment.

FIG. 2 is a detail view of a flow cell and flow cytometer engine of theflow cytometer apparatus of FIG. 1, according to one example embodiment.

FIG. 3 is a flow chart of a method for detecting a clog in a flowcytometer apparatus, according to one example embodiment.

FIG. 4 is a graph showing example event counts over time for a sampledmicroplate, according to one example embodiment.

FIG. 5 is a graph showing an initial calibration of the clog detectionsystem with inter-well fluorescent bead sips with a fixed time intervaland subsequent inter-well fluorescent bead sips measured in real-timewith respect to a user-defined microplate sampling protocol.

FIG. 6 is a graph showing fluorescent bead sip counts over time.

FIG. 7 is a flow chart of a method for clearing a clog from a flowsystem, according to one example embodiment.

DETAILED DESCRIPTION

Example flow cytometer apparatus and methods for detecting and/orclearing of a clog in the flow cytometer apparatus are described herein.Any example embodiment or feature described herein is not necessarily tobe construed as preferred or advantageous over other embodiments orfeatures. The example embodiments described herein are not meant to belimiting. It will be readily understood that certain aspects of thedisclosed methods can be arranged and combined in a wide variety ofdifferent configurations, all of which are contemplated herein.

Furthermore, the particular arrangements shown in the Figures should notbe viewed as limiting. It should be understood that other embodimentsmay include more or less of each element shown in a given Figure.Further, some of the illustrated elements may be combined or omitted.Yet further, an example embodiment may include elements that are notillustrated in the Figures.

As used herein, “about” means +/−5%.

As used herein “well” means structure which contains a sample to beanalyzed, a control or an aliquot of marker particles.

As used herein “microplate” and “plate” refer to a structure capable ofholding one or more samples to be analyzed or aliquot of markerparticles.

As used herein “sample” refers to any quantity of liquid which maycontain particles of interest or marker particles that are detectable bya particle analyzer. More specifically a sample may include a fluidsolution or suspension containing particles of interest or markerparticles to be detected and/or analyzed using a method and/or apparatusdisclosed herein. The particles of interest in a sample may be tagged,such as with a fluorescent tag. The particles of interest may also bebound to a bead, a receptor, or other useful protein or polypeptide, ormay just be present as free particles, such as particles found naturallyin a cell lysate, purified particles from a cell lysate, particles froma tissue culture, etc. The sample may include chemicals, either organicor inorganic, used to produce a reaction with the particles of interest.When the particles of interest are biomaterials, drugs may be added tothe samples to cause a reaction or response in the biomaterialparticles. The chemicals, drugs or other additives may be added to andmixed with the samples when the samples are in sample source wells orthe chemicals, drugs or other additives may be added to the samples inthe fluid flow stream after the samples have been uptaken by theautosampler.

As used herein, the term “biomaterial” refers to any organic materialobtained from an organism, either living or dead. The term “biomaterial”also refers to any synthesized biological material such as synthesizedoligonucleotides, synthesized polypeptides, etc. The synthesizedbiological material may be a synthetic version of a naturally occurringbiological material or a non-naturally occurring biological made fromportions of naturally occurring biological materials, such as a fusionprotein, or two biological materials that have been bound together, suchas an oligonucleotide, such as DNA or RNA, bound to a peptide, eithercovalently or non-covalently, that the oligonucleotide does not normallybind to in nature.

As used herein, the term “oligonucleotide” refers to anyoligonucleotide, including double and single-stranded DNA, RNA, PNAs(peptide nucleic acids) and any sequence of nucleic acids, eithernatural or synthetic, derivatized or underivatized.

As used herein, “peptide” refers to all types of peptides and conjugatedpeptides including: peptides, proteins, polypeptides, protein sequences,amino acid sequences, denatured proteins, antigens, oncogenes andportions of oncogenes.

As used herein, the term “organism” refers not only to animals, plants,bacteria, viruses, etc. but also to cell cultures, reproducedoligonuncleotides, etc. made from organic material obtained fromanimals, plants, bacteria, viruses, etc.

As used herein, the term “drug” refers to any type of substance that iscommonly considered a drug. A drug may be a substance that acts on thecentral nervous system of an individual, eg a narcotic, hallucinogen,barbiturate, or a psychotropic drug. For the purposes of the presentinvention, a drug may also be a substance that kills or inactivatesdisease-causing infectious organisms. In addition, a drug may be asubstance that affects the activity of a specific cell, bodily organ orfunction. A drug may be an organic or inorganic chemical, a biomaterial,etc.

As used herein, an “aliquot” is a sip of a sample taken from a well viaa probe of a flow cytometer.

As used herein, the term “fluidic pathway” or “conduit” refers to devicesuch as a tube, channel, etc. through which a fluid stream flows. Afluidic pathway may be composed of several separate devices, such as anumber of connected or joined pieces of tubing or a single piece oftubing, alone or in combination with channels or other differentdevices. In various embodiments, a fluidic pathway may include any tubethat may be used with a peristaltic pump that has compressioncharacteristics that allow a peristaltic pump to move samples separatedby a separation gas or aliquots of marker particles through the tube ata speed of at least 6 samples per minute without causing adjacentsamples to mix with each other.

As used herein “marker particles” may include control particles, beadsor microbeads and further refers to one or more particles detectable bya flow cytometer system (for example a system as described in U.S. Pat.No. 6,878,556 and WO2010005617) that may uptake from a sample containeran aliquot of a sample suspected of having therein particles of interestto be analyzed.

In one embodiment, introducing the plurality of samples into a conduitor fluidic pathway includes uptaking each of the plurality of samplesfrom a respective sample container. For example, the respective samplecontainer may be a microplate having rows and columns of sample wellsfor holding samples to be tested.

According to one embodiment, fluid gaps are gas gaps, for example airgaps.

According to another embodiment, flowing the plurality of samplesincludes moving the samples with a pump, gravity, acoustic means,microcapillary action, pressurization or any combination thereof.

According to still another embodiment, detecting particles of interestwhen present in the detector zone depends on the optical and/or physicalcharacteristic of interest selected for the particles of interest.According to a further embodiment detecting marker particles may dependon the optical and/or physical characteristics selected for the markerparticles. For example, marker particles may be selected based uponoptical and/or physical characteristics which may be the same ordifferent from the optical and/or physical characteristics of theparticles of interest.

In one embodiment, the system may include a sample to be analyzed whichmay be transported from a sample well to a detector of the flowcytometer via a conduit or fluidic pathway. The sample to be analyzedmay be taken up from the sample well via a probe. In between samples,the probe may uptake a separation gas. Multiple samples may betransported in the conduit sequentially. The multiple samples may beseparated from each other via fluid gaps (e.g. air) and a plurality ofsamples to be analyzed may be moved along the conduit or fluidic pathwayto the detector thereby creating a flowing stream of samples to beanalyzed. Particles of interest within the sample to be analyzed mayflow in a flow cell and pass an illumination source in a detector zone.The demarcation or delineation between the plurality of samples to beanalyzed in the flowing stream within the conduit or fluidic pathway maybe the fluid gap positioned between each one of the plurality of samplesto be analyzed. For example, a first sample to be analyzed is separatedfrom a second sample to be analyzed via one or more air gaps accordingto one embodiment of the present invention.

From a sample to be analyzed, a population of particles may beidentified based upon their optical/physical characteristics such aslight scatter, emission properties, size, but not limited thereto.Particles of interest from the plurality of samples to be analyzedsharing the desired characteristic may be detected by the detector inthe detector zone as the particles of interest pass between the detectorand a light source that provides a light path that strikes the detectorwithin the detector zone (e.g., a laser interrogation point). As thesamples to be analyzed pass the detector (e.g. photomultiplier tube) ofthe particle analyzer, samples having particles of interest with opticaland/or physical characteristics that are within the desired/set opticaland/or physical characteristics will be identified as an event (particlehaving or producing the desired optical and/or physical properties foranalysis). The air gaps between the samples do not contain particles ofinterest that will be recorded as an event. Therefore, a graph of thedata points of fluorescence sensed versus time for a series of samplesanalyzed using a flow cytometer may form distinct groups, each alignedwith the time that a sample containing particles passes through thelaser interrogation or detection point. In order to detect the presenceof each of two or more different types of samples, in a heterogeneousplurality of samples, each of the two or more different types of samplesmay be tagged with different fluorescent tags, different amounts of asingle tag or some combination of different tags and different amount ofa single tag. In such a case, the groupings of data points may varyvertically on a fluorescence versus time graph, depending on which typeof sample is being sensed. As with the case of sensing a single type ofsample, each sensed sample will exhibit a group of data points alignedwith the time that the sample passes through the laser interrogationpoint.

In another embodiment, the marker particles may be comingled with thesample to be analyzed and the apparatus and method may utilize markerparticles to identify the location within the sample stream of a sampleto be analyzed. In an embodiment in which marker particles are notpresent, the delineation of each sample to be analyzed in the samplestream may be easily identified when particles of interest in thesamples to be analyzed are relatively similar in terms of theirconcentration and/or other optical and/or physical characteristics. Ifthe multiple samples to be analyzed in the conduit or fluidic pathwayare different with respect to the particles of interest—for example, ifthere are very few particles of interest in some of the samples to beanalyzed, or if there are large gaps inserted between the air gaps wherea sample to be analyzed would be expected but for an instrumentmalfunction—the location of the sample to be analyzed in the data streammay become problematic in the absence of marker particles.

In one embodiment in which the marker particles are comingled with asample to be analyzed. The flow cytometric properties of the markerparticles may be different from those of the particles of interestwithin the samples to be analyzed. The difference in the optical and/orphysical characteristics of the marker particles along with the factthat there may be known numbers of marker particles comingled with eachsample to be analyzed may allow a user to delineate the location of thesample to be analyzed in the data stream even if there are no particlesof interest in the sample to be tested other than the marker particles.

In various embodiments, marker particles may be added to a wellcontaining sample or a non-sample containing well. The marker particlesmay have a known characteristic such as known size, fluorescentintensity, forward light scatter and side light scatter for example.However, other characteristics that are well known in the art fordetecting and characterizing particles may be useful in a particleanalyzer such as the particle analyzer disclosed in U.S. Pat. No.6,878,556 may also be useful. For example, in one embodiment, the markerparticles may be introduced between samples, and thus demarcate theanticipated beginning location in the flowing stream of a sample to beanalyzed prior to the sample to being analyzed entering the detectorzone of the particle analyzer. Once the bolus of sample to be analyzedmoves past the detector zone, a subsequent bolus of marker particles inthe conduit may move past the detector zone, indicating the anticipatedending location in the flowing stream of a sample to be analyzed. Thesemarker particles have known physical and/or optical characteristics,including emission spectra, intensity, shape, size, which are capturedby the particle analyzer (e.g. flow cytometer). The marker particles maybe added in known positions relative to the samples to be analyzed inthe flowing stream. A fault detection system may then utilize thecharacteristics and the temporal position within the flowing stream ofthe marker particles to determine the anticipated location of a sampleto be analyzed in the flowing stream and/or data stream and to determinewhether there is a clog in the flow cytometer system.

In one embodiment, in which the samples to be analyzed and markerparticles alternate in the conduit, the time boundaries of each sampleto be analyzed when present in the detector zone may be set based on thelowest number of events associated with a histogram developed based uponthe marker particles in a given aliquot. Specifically, boundariesrepresenting time gates may be determined before and after a markerparticle histogram to represent the location for the end point of afirst sample and the starting point of a subsequent second sample,respectively, to be analyzed when present in the detector zone. Thehistograms may be developed in real-time during sample flow and analysisthrough the detector zone to aid in clog detection and sample analysis.In addition, the correlation of a histogram back to the x-y coordinatesof a sample container (for example, the position of a well on a plate)may be determined based upon the timing and sampling order used in thesampling process. Since the samples to be analyzed and marker particlesalternate in the conduit or fluidic pathway, each histogram peak of asample to be analyzed may appear at a unique time period and may beassigned to the sample well identified by the marker particle data.

One advantage of this method may be that in experiments performed withan autosampler sampling system as described herein, there are oftencases when individual wells of the plate may contain no sample eventsdue to sampling error or effects of chemical treatment of the sample.Moreover, it is not typically known in advance which wells may be emptyof test sample events. With this method, wells that contain no testsample events may be accurately identified via the marker particlehistograms.

One embodiment provides delineation between samples to be analyzed whenthe samples to be analyzed are acquired in a flowing stream separated byair gaps, for example. In this embodiment, marker particles may becommingled in the same wells as the sample particles of interest. Forexample, marker particles may represent about 1-2% of the totalparticles in a given sample. As noted above, there may often be caseswhen an individual well of the plate contains no particles and thereforeno events to detect by the particle analyzer due to sampling error oreffects of chemical treatment of the sample. Moreover, it may not beknown in advance which wells will be empty of particles to beanalyzed/events. With this method, wells that contain no test sampleevents can be accurately identified via the marker particle peaks.

In one aspect, the invention provides a flow cytometer apparatus,comprising:

a flow cell having a first end and a second end;

a sample fluidic pathway having a first end and a second end, whereinthe second end of the sample fluidic pathway is coupled to the first endof the flow cell;

a sample probe coupled to the first end of the sample fluidic pathway;

a sample pump in fluid communication with the sample fluidic pathway;

a waste line having a first end and a second end, wherein the first endof the waste line is coupled to the flow cell; and

a waste pump in fluid communication with the waste line.

Referring now to FIGS. 1-2, a flow cytometer apparatus 100 is shownincluding a flow cell 105 having a first end 106 and a second end 107. Asheath fluidic pathway 110 may be coupled to the first end 106 of theflow cell 105 and a sheath pump 115 may be in fluid communication withthe sheath fluidic pathway 110. In one embodiment, the sheath fluidicpathway 110 may be coupled to a sheath fluid reservoir 175 that isconfigured to hold sheath fluid 111. During testing operations performedby the flow cytometer apparatus 100, the sheath pump 115 may drive thesheath fluid 111 from the sheath fluid reservoir 175 through the sheathfluidic pathway 110 and through the flow cell 105.

The flow cytometer apparatus 100 also includes a sample fluidic pathway120 having a first end 121 and a second end 122. The second end 122 ofthe sample fluidic pathway 120 is coupled to the first end 106 of theflow cell 105. The flow cytometer apparatus 100 further includes asample probe 125. An example probe may include a 0.01 inch ID, 1/15 inchOD stainless steel needle compatible with HPLC ferrule fittings. In oneembodiment, in order to reduce carryover of samples between wells, theprobe 125 may have a conical tip. In another embodiment, silicone orother hydrophobic agent may coat the tip of the sampling probe 125 tohelp minimize sample carryover. In an alternative embodiment, the entireprobe 125 may be made of a hydrophobic material to reduce carryover.Suitable hydrophobic materials for use in the coating or for making theentire hydrophobic probe include: Teflon® (poly(tetrafluoroethylene)(PTFE)), Kynar® (polyvinylidene fluoride), Tefzel®(ethylene-tetrafluoroethylene copolymer),tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer resin (PFA), atetrafluoroethylene-hexafluoropropylene copolymer (EFP), polyether etherketone (PEEK), etc.

In one embodiment, the sample probe 125 may be coupled to a probe holder126. In one embodiment, the flow cytometry apparatus 100 may include aconventional autosampler 127, such as the Gilson 215 liquid manager. Theprobe holder 126 of the autosampler 127 may take the form of anadjustable arm. As probe holder 126 moves side to side and up and down,probe 125 is lowered into individual source wells 186 of a well plate185 to obtain a sample that has been tagged with a fluorescent tag to beanalyzed using the flow cytometry apparatus 100. In turn, the sampleprobe 125 is coupled to the first end 121 of the sample fluidic pathway120, and a sample pump 130 is in fluid communication with the samplefluidic pathway 120. In operation, the sample probe 125 may take up asample from a well in a well-plate, for example, and then advance thesample into the sample fluidic pathway 120. The sample pump 130 may thendrive a sample stream 123 through the sample fluidic pathway 120 to asample inlet port 108 and through the flow cell 105. In one embodiment,the flow cell 105 may include a laser interrogation device that mayexamine individual samples flowing from flow cell 105 at a laserinterrogation point. Once the sample stream 123 enters the flow cell105, then the sheath fluid 111 flowing through the flow cell 105 may aidin hydrodynamic focusing of the samples in the center of the flow cell105. As shown, the sample stream 123 may include a series of sampleseach separated by an air gap. These air gaps may be formed by allowingsample probe 125 to intake air in between intaking sample material fromeach of sample wells 186.

In addition, the flow cytometer apparatus 100 includes a waste line 135having a first end 136 and a second end 137. The first end 136 of thewaste line 135 is coupled to the second end 107 of the flow cell 105 inorder to receive waste fluid 138 (i.e., the combination of sheath fluidand the sample stream 123) upon exiting the flow cell 105. A waste pump140 is in fluid communication with the waste line 135 to help advancethe fluid 138 through the waste lines 135 and into a waste fluidreservoir 170 coupled to the second end 137 of the waste line 135.

In one embodiment, the sheath pump 115, the sample pump 130 and/or thewaste pump 140 may be various conventional peristaltic pumps. Oneexample peristaltic pump is Gilson Minipuls 3. In one embodiment, theperistaltic pumps may be operated in a manner that reduces pulsatileflow, thereby improving the sample characteristics in the flowcytometer. For example, a tubing length greater than 20 inches betweenpump and flow cytometer may be used or a linear peristaltic pump such asthe Digicare LP5100 may be used to improve the sample characteristics.In one embodiment, the sample fluidic pathway 120, the sheath fluidicpathway 110 and/or the waste line 135 may be made of an elastomertubing, such as nitrile (NBR), Hypalon, Viton, silicone, polyvinylchloride (“PVC”), Ethylene-Propylene-Diene-Monomer (“EPDM”),EPDM+polypropylene, polyurethane or natural rubber, among otherpossibilities. An example of such a tube may be a polyvinyl chloride(PVC) tube having an inner diameter of about 0.01 to 0.03 inches and awall thickness of about 0.01 to 0.03 inches. In one embodiment, apreferred tube for a fluidic pathway may be a PVC tube having an innerdiameter of about 0.02 inches and a wall thickness of about 0.02 inches.

In addition, the flow cytometer apparatus 100 may include a three-portvalve 145 that may be coupled to the waste line 135 between the flowcell 105 and the waste pump 140. The three-port valve 145 may have afirst port 146, a second port 147 and a third port 148. The first port146 of the three-port valve 145 may be arranged in series with thesecond port 147 such that the first-port 146 is arranged closer to theflow cell 105 than the second port 147. In turn, the third port 148 ofthe three-port valve 145 may be configured to communicate withatmosphere 149. In normal operation, the third port 148 is closed andwaste fluid 138 flows freely through the valve between the first port146 and the second port 147. As described in more detail with respect tothe third aspect of the invention, the third port 148 may be opened upona detection of a clog to aid in the method for clearing the clog fromthe flow cytometer apparatus 100.

In one embodiment, the flow cytometer 100 may include a decontaminationsolution reservoir 150, a cleaning solution reservoir 155 and acell-compatible fluid reservoir 160 each configured to receive at leasta tip of the sample probe 125. Example decontamination solutions thatmay be provided in the decontamination solution reservoir 150, mayinclude, but are not limited to, a 5.25% concentration of SodiumHypochlorite to water. Example cleaning solutions that may be providedin the cleaning solution reservoir 155, may include, but are not limitedto a 1.5% concentration of Citranox to water.

Further, deionized water may be provided in the cell-compatible fluidreservoir 160. Example cell-compatible fluids that may be provided inreservoir 160 include deionized water, a salt water solution, a buffer,or any other solution with cell-neutral properties. The effect of eachof these solutions is discussed in detail with respect to the thirdaspect of the invention. In a further embodiment, the flow cytometer 100may also include a back flush waste reservoir 165 configured to receiveat least the tip of the sample probe 125. The back flush waste reservoir165 may receive waste fluids pumped through the flow cytometer apparatusin a reverse-mode as part of a method for clearing a clog from thesystem. In one embodiment, each of the foregoing reservoirs 150, 155,160, 165 may be a well that is defined in a well-plate 180. Thiswell-plate 180 may be the same as or different than the well-plate 185in which the test samples are placed.

FIG. 3 is a flow chart of a method 300 for detecting a clog in a flowcytometer, according to one example embodiment. Example methods, such asmethod 300 of FIG. 3 and method 700 of FIG. 7, may be carried out by anoperator or a control system, including the fault detection system. Inoperation in one example embodiment, high-throughput flow cytometry mayuse a pump system to fill a sample fluidic pathway 120 with a stream 123of discrete sample particle suspensions aspirated from one or more wellsof a microplate and separated from each other by air gaps. The entiresample stream 123 may be continuously delivered to the flow cell 105 topermit data from each of the samples in the microplate 185 to beacquired and stored in a single data file. During operation, the flow ofthe sample stream 123 may become interrupted by a clog.

As such, in a second aspect, the invention provides a method 300 fordetecting a clog in a flow cytometer apparatus of a type known in theart or in a flow cytometer apparatus 100 according to the first aspectof the invention. As shown by block 305, method 300 involves a faultdetection system of a flow cytometer 100 detecting a first plurality ofevents associated with a first aliquot from a first sample well. As usedherein, an “event” refers to the presence of one or more particles ofinterest or marker particles associated with an aliquot of a sample,where the presence may be indicated by physical or chemical features,such as fluorescent intensity of the samples. Then, at block 310, thefault detection system determines a count of the first plurality ofevents associated with the first aliquot. As used herein, “count”literally refers the number of events detected for a given aliquot of asample. FIG. 4 reflects data from a typical microplate sample set thatprovides an example of a plurality of events plotted against time duringthe course of sampling and data acquisition operations of the flowcytometer apparatus 100. The spikes in the graph represent individualsample wells and the larger low event count gaps between a microplaterow or column represents a microplate shake and/or a sample probe rinse.Next, at block 315, the fault detection system determines whether thecount of the first plurality of events is below a minimum counttolerance. And, if the count of the first plurality of events is belowthe minimum count tolerance, then at block 320 the fault detectionsystem determines that the flow cytometer has a clog. If the count ofthe first plurality of events is equal to or above the minimum counttolerance, then at block 325 the fault detection system detects a secondplurality of events associated with a second aliquot from a secondsample well thereby continuing a normal sampling operation.

In one embodiment, the fault detection system may include a controlsystem that may take the form of program instructions stored on anon-transitory computer readable medium and a processor that executesthe instructions. However, a control system may take other formsincluding software, hardware, and/or firmware. The fault detectionsystem may be part of or include the data processing system of the flowcytometer or the fault detection system may be a processor that isseparate from that of the flow cytometer.

In one embodiment, method 300 may further involve the fault detectionsystem determining a count of the second plurality of events. Then thefault detection system may determine whether the count of the secondplurality of events is below a minimum count tolerance. And, if thecount of the second plurality of events is below the minimum counttolerance, the fault detection system then may determine that the flowcytometer has a clog. On the other hand, if the count of the secondplurality of events is equal to or above the minimum count tolerance,then the fault detection system may detect a third plurality of eventsassociated with a third aliquot from a third sample well of the flowcytometer thereby continuing a normal sampling operation.

In one embodiment, the first aliquot may include a plurality ofparticles of interest such that the first plurality of countscorresponds to the plurality of particles of interest.

In one embodiment, the count tolerance may be a minimum of fifteenevents per second. In various embodiments, the sampling protocol may beanalyzed to determine the longest period of time the sample probe may beimmersed in one of the decontamination, cleaning or cell-compatiblefluid reservoirs during the plate sampling operation. This informationmay then be entered into the fault detection system so that the faultdetection system sets the longest time period during which the faultdetection system may expect event counts to be below fifteen events persecond. In one embodiment, an additional three seconds may be added tothis immersed time period. If the sampling protocol does not include anyprobe rinses or well-plate shaking, then the longest time period duringwhich the fault detection system may expect event counts to be belowfifteen events per second is defaulted to five seconds.

In a further embodiment, before determining whether the count of thefirst plurality of events is below a minimum count tolerance, the faultdetection system may detect at least 1500 total events from one or morealiquots of samples from one or more sample wells. This may permit thefault detection system to gather enough data to calibrate the system andto determine time gates discussed in more detail below. In oneembodiment, as shown in FIG. 5, an example initial calibrationfluorescent bead sip via the sample probe 125 may be performed over afixed time interval to determine the fluorescent intensity and eventcount for this sample run. If this initial calibration sip is determinedto be below a count tolerance, then the flow cytometer apparatus may bedetermined to be clogged. If this initial calibration sip is above thecount tolerance, then it may be used as a measurement against subsequentbead sips as shown in FIGS. 5 and 6. For example, FIG. 6 shows exampledata gathered via the fault detection system, monitoring the frequencyand event counts for subsequent bead sips via the sample probe 125 inreal-time. In one embodiment, the fault detection system is set toexpect that the first fluorescent bead sip will be a continuous peakthat is least two seconds long. The fault detection system may recordthe event count of this initial peak and then the fault detection systemmay determine that future between-well fluorescent bead sips areapproximately a half second long and may have an event count that isdefined as((CalibrationSipEventCount*BeadSipTime)/CalibrationSipTime)*0.35. If abead sip does not meet the sip duration time of a half second or achievean event count within the expected time duration based on the samplingtime duration plus up to two additional seconds, the bead sip may bemarked as a missed sip and a potential clog. The fault detection systemmay be programmed to expect a regular interval of bead sips betweenwells when the system is sampling a plate without a clog. Therefore, inthis embodiment, if a bead sip is missed then the flow cytometer systemis determined to be in a clogged state.

In another embodiment, the first aliquot may include a plurality ofmarker particles such that the first plurality of counts corresponds tothe plurality of marker particles of the first aliquot. In addition, thesecond aliquot may include a plurality of particles of interest suchthat the second plurality of counts corresponds to the plurality ofparticles of interest. And, the third aliquot may include a plurality ofmarker particles such that a third plurality of counts corresponds tothe plurality of marker particles of the third aliquot. This arrangementmay permit time gates to be established as described in more detailbelow. In this embodiment, every other well of a well-plate may befilled with samples containing marker particles and the wellstherebetween may be filled with samples containing particles ofinterest.

In another embodiment, method 300 may further involve the faultdetection system determining a beginning of a count interval for thesecond aliquot. Then the fault detection system may determine an end ofthe count interval for the second aliquot. And the fault detectionsystem may detect the plurality of events associated with the secondaliquot during the count interval. This embodiment establishes a windowof time in which the fault detection system expects to detect particles.In one embodiment, the step of determining a beginning of a countinterval for the second aliquot may include the fault detection systemdetermining data corresponding to a first histogram. This firsthistogram may be based upon the first plurality of events detected forthe plurality of marker particles of the first aliquot over time. Thenthe fault detection system may determine a first time gate and a secondtime gate, such that the first time gate corresponds to an earliestdetected event in the first histogram and the second time gatecorresponds to a latest detected event in the first histogram. Thesecond time gate may then be established as the beginning of the countinterval. In another embodiment, the step of determining an end of thecount interval for the second aliquot may include the fault detectionsystem determining data corresponding to a second histogram. This secondhistogram may be based upon the third plurality of events detected forthe plurality of marker particles of the third aliquot over time. Next,the fault detection system may determine a third time gate and a fourthtime gate, such that the third time gate corresponds to an earliestdetected event in the second histogram and the fourth time gatecorresponds to a latest detected event in the second histogram. Thethird time gate may then be established as the end of the countinterval. In a further embodiment, the fourth time gate may beestablished as the beginning of a second count interval for a fourthaliquot.

In one embodiment, method 300 may further include the fault detectionsystem determining whether the count of the first plurality of events isabove a maximum count tolerance. And, if the count of the firstplurality of events is above a maximum count tolerance, then the faultdetection system may determine that there is a system anomaly in theflow cytometer. In response to determining the presence of a systemanomaly, the fault detection system may pause the sampling operation ofthe flow cytometer. If, however, the count of the first plurality ofevents is equal to or below a maximum count tolerance, then the faultdetection system detects the second plurality of events associated withthe second aliquot from the second sample well of the flow cytometer.

In one embodiment, the step of the fault detection system determiningthat the flow cytometer has a clog may include the fault detectionsystem detecting at least one more plurality of events associated with asubsequent aliquot in a fluidic pathway of the flow cytometer. Then thefault detection system may determine whether the count of the at leastone more plurality of events is below the minimum count tolerance. Ifthe count of the at least one more plurality of events is below theminimum count tolerance, then the fault detection system may pause asampling operation of the flow cytometer. On the other hand, if thecount of the first plurality of events is equal to or above the minimumcount tolerance, then the sampling operation of the flow cytometer maycontinue. This embodiment may permits the flow cytometer system tocontinue the sampling operation for a certain amount of time afterdetermining a count is below the count tolerance in order to confirmthat the low count was not due to a sampling error or due to a chemicalreaction diminishing the number of particles present in a given sample.

In another embodiment, the method 300 may further include the faultdetection system detecting a fourth plurality of events associated witha fourth aliquot from a fourth sample well of the flow cytometer. Thenthe fault detection system may determine a count of the fourth pluralityof events. Next, the fault detection system may determine whether thecount of the fourth plurality of events is below a minimum counttolerance. If the count of the fourth plurality of events is below theminimum count tolerance, then the fault detection system may determinewhether the counts of the first, second and third plurality of eventswere dropped over time such that the counts trended downward. In thiscase, if the counts of the first, second and third plurality of eventsdropped over time, then the fault detection system may determine thatthe flow cytometer has a clog and then may pause a sampling operation ofthe flow cytometer. Alternatively, if the counts of the first, secondand third plurality of events did not drop over time, then the faultdetection system may continue the sampling operation of the flowcytometer for at least one more aliquot. In this embodiment, the faultdetection system is allowed to assess whether a downward trend in countswas present leading up to the count that did not meet the counttolerance. This may have the benefit of preventing the sample probe fromtaking up further samples that would go to waste if clog remediationsteps are undertaken.

In one embodiment, the first plurality of events may include a pluralityof events related to fluorescence of a plurality of particles in thefirst aliquot. In a further embodiment, the plurality of particles inthe first aliquot may be configured to fluoresce in the presence oflight of a predetermined frequency. In this embodiment, the flowcytometer may include a laser configured to emit light of thepredetermined frequency. The flow cytometer may also be configured toshine light emitted from the laser on the plurality of particles. Thenthe step of detecting a plurality of events may include detecting anevent related to fluorescence of the plurality of particles. In variousother embodiments, other events that may be detected are cellular,chemical and protein aggregates or debris.

In one embodiment, method 300 may further include the provision of aflow cytometer system according to the first aspect of the invention.Then, in response to determining that the flow cytometer has a clog, thefault detection system may activate the waste pump, thereby applyingnegative pressure to one or more of a waste line, a flow cell and asample fluidic pathway. And a sample pump may also be activated by thefault detection system. Then the flow cytometer system may cycle thesample probe into and out of a decontamination solution reservoir suchthat a decontamination fluid may be driven by the sample pump throughone or more of the flow cell, the sample fluidic pathway and the wasteline, thereby clearing a clog.

In a further embodiment, the flow cytometer system may cycle the sampleprobe into and out of a cleaning solution reservoir. Then the samplepump may drive a cleaning fluid through one or more of the flow cell,the sample fluidic pathway or the waste line, thereby cleaning away thedecontamination fluid.

In a still further embodiment, the method 300 may also include the flowcytometer system cycling the sample probe into and out of acell-compatible fluid reservoir. Then sample pump may drive acell-compatible fluid through one or more of the waste line, the flowcell and the sample fluidic pathway, thereby removing any remainingcleaning fluid.

In another embodiment, the method 300 may further include the provisionof a sheath fluidic pathway coupled to the first end of the flow celland a sheath pump in fluid communication with the sheath fluidicpathway. The flow cytometer system may activate the sheath pump, whichmay then drive a sheath fluid through one or more of the sheath fluidicpathway, the flow cell and the waste line. In a third aspect, theinvention provides a method for clearing a clog from the flow cytometerapparatus 100 according to the first aspect of the invention. As shownin FIG. 7, at block 705, a flow cytometer apparatus 100 is providedaccording to the first aspect of the invention. Then, at block 710, thewaste pump 140 is activated to apply negative pressure to one or more ofthe waste line 135, the flow cell 105 and the sample fluidic pathway120. The sample pump 130 is activated, at block 715. At block 720, thesample probe 125 cycles into and out of a decontamination solutionreservoir 150 and drives a decontamination fluid, via the sample pump130, through one or more of the flow cell 105, the sample fluidicpathway 120 and the waste line 135, thereby clearing a clog.

In a further embodiment, method 700 may include the sample probe 125cycling into and out of a cleaning solution reservoir 155. Then acleaning fluid may be driven via the sample pump 130, through one ormore of the flow cell 105, the sample fluidic pathway 120 or the wasteline 135, thereby cleaning away the decontamination fluid.

In another embodiment, method 700 may include the sample probe 125 maycycle into and out of a cell-compatible fluid reservoir 160. Then acell-compatible fluid may be driven through one or more of the wasteline 135, the flow cell 105 and the sample fluidic pathway 120, therebyremoving any remaining cleaning fluid.

In one embodiment, the method 700 may further include activating thesheath pump 115 and driving a sheath fluid 111 through one or more ofthe sheath fluidic pathway 110, the flow cell 105 and the waste line135. The flowing sheath fluid 111 may further aid in flushing the clog,cleaning and/or removing cleaning solution from the flow cell 105 and/orwaste line 135 (and/or the sample fluidic pathway 120, if the flowcytometer apparatus 100 is operated in reverse mode).

In one embodiment, cycling of the sample probe 125 into and out of thedecontamination solution reservoir 150 may occur for a period of timeranging from about two minutes to about five minutes. In a furtherembodiment, each cycle of the sample probe 125 into and out of thedecontamination solution reservoir 150 may involve placing at least thetip of the sample probe 125 into the decontamination solution reservoir150 for at least one second and, more preferably, between about onesecond to about 5 seconds and removing the tip of the sample probe 125from the decontamination solution reservoir 150 for at least one secondand, more preferably, between about one second to about 5 seconds. Inanother embodiment, the method 700 may include pumping a plurality ofdecontamination fluid samples separated by air samples through thesample fluidic pathway 120 during the cycling of the sample probe 125into and out of the decontamination solution reservoir 150. The lengthor amount of the decontamination fluid samples and the air samples aredetermined by the amount of time the sample probe spends immersed indecontamination fluid and immersed in atmosphere, respectively. Theprovision of air samples (i.e. air gaps) in between samples ofdecontamination fluid proved more effective than continuously pullingdecontamination fluid into the sample fluidic pathway 120. The sameresults are applicable for the cycling of the cleaning solution and thedeionized water.

In a further embodiment, the method 700 may also include pumping aplurality of cleaning fluid samples separated by air samples through atleast the sample fluidic pathway 120 during the cycling of the sampleprobe 120 into and out of the cleaning solution reservoir 155. Inanother embodiment, the sample probe 125 may be cycled into and out ofthe cleaning solution reservoir 155 for a period of time ranging fromabout two minutes to about five minutes. In a further embodiment, eachcycle of the sample probe 125 into and out of the cleaning solutionreservoir 155 may involve placing at least the tip of the sample probe125 into the cleaning solution reservoir 155 for about one second toabout 5 seconds and removing the tip of the sample probe 125 from thecleaning solution reservoir 155 for about one second to about 5 seconds.In a still further embodiment, the sample probe 125 may be cycled intoand out of the cleaning solution reservoir 155 at least 30 times.

In another embodiment, method 700 may include pumping a plurality ofdeionized water samples separated by air samples through at least thesample fluidic pathway 120 during the cycling of the sample probe 125into and out of the deionized water reservoir 160. In anotherembodiment, the sample probe 125 may be cycled into and out of thedeionized water reservoir 160 for a period of time ranging from abouttwo minutes to about five minutes. In a further embodiment, each cycleof the sample probe 125 into and out of the deionized water reservoir160 may involve placing at least the tip of the sample probe 125 intothe cell-compatible fluid reservoir 160 for at least one second and,more preferably, between about one second to about 5 seconds andremoving the tip of the sample probe 125 from the deionized waterreservoir 160 for at least one second and, more preferably, betweenabout one second to about 5 seconds. In a still further embodiment, thesample probe 125 may be cycled into and out of the cell-compatible fluidreservoir 160 at least 30 times.

In one embodiment, prior to activating the waste pump 140, a samplingoperation and a data acquisition operation may be ceased. The samplingand data acquisition operations may be ceased in response to a faultdetection system determining a clog is present in the flow cytometerapparatus, for example.

In one embodiment, cycling the sample probe 125 into and out of thedecontamination solution reservoir 150 may involve: (a) operating theflow cytometer system 100 in a forward mode, (b) deactivating the wastepump 140 and the sample pump 130, (c) holding a plurality ofdecontamination fluid samples separated by air samples in at least thesample fluidic pathway 120, (d) closing the second port 147 of thethree-port valve 145 and opening the third port 148 of the three-portvalve 145, (e) operating the sample pump 130 in a reverse-mode andflowing the plurality of decontamination fluid samples in reversethrough at least the sample fluidic pathway 120. Operating in reversemode may be helpful in dislodging a clog if the clog does not clearwhile operating in a forward mode. In addition, holding thedecontamination fluid in contact with the clog for a period of time mayhelp breakdown the clog down.

In another embodiment, cycling the sample probe 125 into and out of thedecontamination solution reservoir 150 may involve: (a) operating theflow cytometer system 100 in a forward mode, (b) deactivating the wastepump 140 and the sample pump 130, (c) holding a plurality ofdecontamination fluid samples separated by air samples in at least thesample fluidic pathway 120, (d) operating the sample pump 130 in theforward-mode and flowing the plurality of decontamination fluid samplesthrough the three-port valve 145 and into the waste reservoir 170.

In one embodiment, cycling the sample probe 125 into and out of thecleaning solution reservoir 155 may involve: (a) operating the flowcytometer system 100 in the forward mode, (b) deactivating the wastepump 140 and the sample pump 130, (c) holding a plurality of cleaningfluid samples separated by air samples in the sample fluidic pathway120, (d) closing the second port 147 of the three-port valve 145 andopening the third port 148 of the three-port valve 145 (e) operating thesample pump 130 in the reverse-mode and flowing the plurality ofdecontamination fluid samples in reverse through at least the samplefluidic pathway 120.

In another embodiment, cycling the sample probe 125 into and out of thecleaning solution reservoir 155 may involve: (a) operating the flowcytometer system 100 in the forward mode, (b) deactivating the wastepump 140 and the sample pump 130, (c) holding a plurality of cleaningfluid samples separated by air samples in the sample fluidic pathway120, (d) operating the sample pump 130 in the forward-mode and flowingthe plurality of decontamination fluid samples through the three-portvalve 145 and into the waste reservoir 170.

In a still further embodiment, cycling the sample probe 125 into and outof the cell-compatible fluid reservoir 160 may involve: (a) activatingthe sheath pump 115 and (b) operating the flow cytometer system 100 inthe forward mode. This may have the benefit of flushing anydecontamination fluid or cleaning fluid that may have backed up into thesheath fluidic pathway 110.

In another embodiment, the sample probe 125 may be positioned over aback flush waste fluid reservoir 165 when the sample pump 130 isoperating in a reverse-mode. In operation, the back flush waste fluidreservoir 165 may receive decontamination fluid, cleaning solutionand/or cell-compatible fluid after theses fluids have been flushedthrough various components of the flow cytometer 100 as described in theforegoing embodiments.

The above detailed description describes various features and functionsof the disclosed flow cytometer apparatus and methods for detectingand/or clearing of a clog in the flow cytometer apparatus with referenceto the accompanying figures. While various aspects and embodiments havebeen disclosed herein, other aspects and embodiments will be apparent tothose skilled in the art. The various aspects and embodiments disclosedherein are for purposes of illustration and are not intended to belimiting, with the true scope and spirit being indicated by thefollowing claims.

All embodiments of the flow cytometer apparatus may be used in themethods of the second and third aspects of the invention. Note that anyof the foregoing embodiments of any aspect may be combined together topractice the claimed invention unless the context dictates otherwise.

The invention claimed is:
 1. A method, comprising: providing a flowcytometer system comprising (a) a flow cell having a first end and asecond end, (b) a sample fluidic pathway having a first end and a secondend, wherein the second end of the sample fluidic pathway is coupled tothe first end of the flow cell, (c) a sample probe coupled to the firstend of the sample fluidic pathway, (d) a sample pump in fluidcommunication with the sample fluidic pathway, (e) a waste line having afirst end and a second end, wherein the first end of the waste line iscoupled to the flow cell, and (f) a waste pump in fluid communicationwith the waste line; activating the waste pump to apply negativepressure to one or more of the waste line, the flow cell, and the samplefluidic pathway; activating the sample pump; and cycling the sampleprobe into and out of a decontamination solution reservoir and driving adecontamination fluid, via the sample pump, through one or more of theflow cell, the sample fluidic pathway and the waste line, therebyclearing a clog.
 2. The method of claim 1, further comprising: cyclingthe sample probe into and out of a cleaning solution reservoir; anddriving a cleaning fluid, via the sample pump, through one or more ofthe flow cell, the sample fluidic pathway or the waste line, therebycleaning away the decontamination fluid.
 3. The method of claim 2,further comprising: cycling the sample probe into and out of acell-compatible fluid reservoir; and driving cell-compatible fluidthrough one or more of the waste line, the flow cell and the samplefluidic pathway, thereby removing any remaining cleaning fluid.
 4. Themethod of claim 3, further comprising: during the cycling of the sampleprobe into and out of the cell-compatible fluid reservoir, pumping aplurality of cell-compatible fluid samples separated by air samplesthrough at least the sample fluidic pathway.
 5. The method of claim 3,wherein cycling the sample probe into and out of the cell-compatiblefluid reservoir occurs for a period of time ranging from about twominutes to about five minutes.
 6. The method of claim 2, furthercomprising: during the cycling the sample probe into and out of thecleaning solution reservoir, pumping a plurality of cleaning fluidsamples separated by air samples through at least the sample fluidicpathway.
 7. The method of claim 1, further comprising: providing asheath fluidic pathway coupled to the first end of the flow cell and asheath pump in fluid communication with the sheath fluidic pathway;activating the sheath pump; and driving a sheath fluid through one ormore of the sheath fluidic pathway, the flow cell and the waste line. 8.The method of claim 1, further comprising: during the cycling of thesample probe into and out of the decontamination solution reservoir,pumping a plurality of decontamination fluid samples separated by airsamples through the sample fluidic pathway.
 9. The method of claim 1,wherein cycling the sample probe into and out of the decontaminationsolution reservoir occurs for a period of time ranging from about twominutes to about five minutes.
 10. The method of claim 1, furthercomprising: providing a three-port valve coupled to the waste linebetween the flow cell and the waste pump, wherein the three-port valvehas a first port, a second port and a third port, wherein the first portof the three-port valve is arranged in series with the second port ofthe three-port valve such that the first port is arranged closer to theflow cell than the second port, and wherein the third port of thethree-port valve is configured to communicate with atmosphere.
 11. Themethod of claim 10, wherein cycling the sample probe into and out of thedecontamination solution reservoir comprises: (a) operating the flowcytometer system in a forward mode; (b) deactivating the waste pump andthe sample pump; (c) holding a plurality of decontamination fluidsamples separated by air samples in at least the sample fluidic pathway;(d) closing the second port of the three-port valve and opening thethird port of the three-port valve; and (e) operating the sample pump ina reverse-mode and flowing the plurality of decontamination fluidsamples in reverse through at least the sample fluidic pathway.
 12. Themethod of claim 10, wherein cycling the sample probe into and out of thedecontamination solution reservoir comprises: (a) operating the flowcytometer system in a forward mode; (b) deactivating the waste pump andthe sample pump; (c) holding a plurality of decontamination fluidsamples separated by air samples in at least the sample fluidic pathway;and (d) operating the sample pump in the forward mode and flowing theplurality of decontamination fluid samples through the three-port valveand into a waste reservoir.
 13. The method of claim 12, furthercomprising cycling the sample probe into and out of a cleaning solutionreservoir, wherein cycling the sample probe into and out of the cleaningsolution reservoir comprises: (a) operating the flow cytometer system inthe forward mode; (b) deactivating the waste pump and the sample pump;(c) holding a plurality of cleaning fluid samples separated by airsamples in the sample fluidic pathway; (d) closing the second port ofthe three-port valve and opening the third port of the three-port valve;and (e) operating the sample pump in a reverse mode and flowing theplurality of decontamination fluid samples in reverse through at leastthe sample fluidic pathway.
 14. The method of claim 13, furthercomprising cycling the sample probe into and out of a cell-compatiblefluid reservoir, wherein cycling the sample probe into and out of thecell-compatible fluid reservoir comprises: (a) activating a sheath pump;and (b) operating the flow cytometer system in the forward mode.
 15. Themethod of claim 10, further comprising cycling the sample probe into andout of a cleaning solution reservoir, wherein cycling the sample probeinto and out of the cleaning solution reservoir comprises: (a) operatingthe flow cytometer system in a forward mode; (b) deactivating the wastepump and the sample pump; (c) holding a plurality of cleaning fluidsamples separated by air samples in the sample fluidic pathway; and (d)operating the sample pump in the forward mode and flowing a plurality ofdecontamination fluid samples through the three-port valve and into awaste reservoir.
 16. A method, comprising: providing a flow cytometersystem comprising (a) a flow cell having a first end and a second end,(b) a first fluidic pathway having a first end and a second end, whereinthe second end of the first fluidic pathway is coupled to the first endof the flow cell, (c) a sample probe coupled to the first end of thefirst fluidic pathway, (d) a second fluidic pathway having a first endand a second end, wherein the first end of the second fluidic pathway iscoupled to the flow cell, and (e) a pump in fluid communication with thesecond fluidic pathway; activating the pump to apply negative pressureto one or more of the first fluidic pathway, the flow cell, and thesecond fluidic pathway; and cycling the sample probe into and out of adecontamination solution reservoir and driving a decontamination fluid,via the pump, through one or more of the flow cell, the first fluidicpathway, and the second fluidic pathway, thereby clearing a clog.
 17. Anon-transitory computer-readable medium having stored thereoninstructions that, when executed by one or more processors of acomputing device, cause the computing device to perform functions of aflow cytometer system, wherein the flow cytometer system comprises (a) aflow cell having a first end and a second end, (b) a sample fluidicpathway having a first end and a second end, wherein the second end ofthe sample fluidic pathway is coupled to the first end of the flow cell,(c) a sample probe coupled to the first end of the sample fluidicpathway, (d) a sample pump in fluid communication with the samplefluidic pathway, (e) a waste line having a first end and a second end,wherein the first end of the waste line is coupled to the flow cell, and(f) a waste pump in fluid communication with the waste line, thefunctions comprising: activating the waste pump to apply negativepressure to one or more of the waste line, the flow cell, and the samplefluidic pathway; activating the sample pump; and cycling the sampleprobe into and out of a decontamination solution reservoir and driving adecontamination fluid, via the sample pump, through one or more of theflow cell, the sample fluidic pathway and the waste line, therebyclearing a clog.
 18. The non-transitory computer-readable medium ofclaim 17, wherein the functions further comprise: cycling the sampleprobe into and out of a cleaning solution reservoir; and driving acleaning fluid, via the sample pump, through one or more of the flowcell, the sample fluidic pathway or the waste line, thereby cleaningaway the decontamination fluid.
 19. The non-transitory computer-readablemedium of claim 18, wherein the functions further comprise: cycling thesample probe into and out of a cell-compatible fluid reservoir; anddriving cell-compatible fluid through one or more of the waste line, theflow cell and the sample fluidic pathway, thereby removing any remainingcleaning fluid.
 20. The non-transitory computer-readable medium of claim19, wherein the functions further comprise: during the cycling of thesample probe into and out of the cell-compatible fluid reservoir,pumping a plurality of cell-compatible fluid samples separated by airsamples through at least the sample fluidic pathway.
 21. A flowcytometer apparatus, comprising: a flow cell having a first end and asecond end; a first fluidic pathway having a first end and a second end,wherein the second end of the first fluidic pathway is coupled to thefirst end of the flow cell; a sample probe coupled to the first end ofthe first fluidic pathway; a second fluidic pathway having a first endand a second end, wherein the first end of the second fluidic pathway iscoupled to the second end of the flow cell; a pump in fluidcommunication with the second fluidic pathway; and a fault detectionsystem configured to detect a dog in the flow cytometer apparatus,wherein the fault detection system comprises non-transitorycomputer-readable medium having stored thereon instructions that, whenexecuted by one or more processors, cause the fault detection system toperform functions comprising: detecting E first plurality of eventsassociated with a first aliquot from a first sample well; and inresponse to a determination that a count of the first plurality ofevents is below a minimum count tolerance, determining that the flowcytometer apparatus is clogged.
 22. The flow cytometer apparatus ofclaim 21, wherein the functions further comprise: if the count of thefirst plurality of events is equal to or above the minimum counttolerance, then detect a second plurality of events associated with asecond aliquot from a second sample well.
 23. The flow cytometerapparatus of claim 22, wherein the functions further comprise:determining a count of the second plurality of events; determiningwhether the count of the second plurality of events is below a minimumcount, tolerance; if the count a the second plurality of events is belowthe minimum count tolerance, then determining that the flow cytometerapparatus has a clog; and if the count of the second plurality of eventsis equal to or above the minimum count tolerance, then detecting a thirdplurality of events associated with a third aliquot from a third samplewell of the flow cytometer apparatus.
 24. The flow cytometer apparatusof claim 22, wherein, in response to detecting the clog in the flowcytometer apparatus, the functions further comprise: activating the pumpto apply negative pressure to one or more of the first fluidic pathway,the flow cell, and the second fluidic pathway; cycling the sample probeinto and out of a decontamination solution reservoir; and driving adecontamination fluid, via the pump, through one or more of the flowcell, the first fluidic pathway, and the second fluidic pathway, therebyclearing the clog.
 25. The flow cytometer apparatus of claim 21, furthercomprising: a probe holder coupled to the sample probe; and anautosampler coupled to the probe holder.
 26. The flow cytometerapparatus of claim 25, wherein the probe holder of the autosamplercomprises an adjustable arm configured to move the sample probe in atransverse direction and a lateral direction.
 27. The flow cytometerapparatus of claim 21, wherein the flow cell further includes a laserinterrogation device configured to examine individual samples flowingfrom the flow cell at a laser interrogation point.
 28. The flowcytometer apparatus of claim 21, further comprising: a three-port valvecoupled to the second fluidic pathway between the flow cell and thepump, wherein the three-port valve has a first port, a second port and athird port, wherein the first port of the three-port valve is arrangedin series with the second port of the three-port valve such that thefirst port is arranged closer to the flow cell than the second port, andwherein the third port of the three-port valve is configured tocommunicate with atmosphere.
 29. The flow cytometer apparatus of claim21, further comprising: a decontamination solution reservoir configuredto receive at least a tip of the sample probe; a cleaning solutionreservoir configured to receive at least the tip of the sample probe;and a cell-compatible fluid reservoir configured to receive at least thetip of the sample probe.
 30. The flow cytometer apparatus of claim 21,further comprising: a back flush waste reservoir configured to receiveat least a tip of the sample probe.
 31. The flow cytometer apparatus ofclaim 21, further comprising: a waste fluid reservoir coupled to thesecond end of the second fluidic pathway.
 32. The flow cytometerapparatus of claim 21, wherein the sample probe has a conical tip. 33.The flow cytometer apparatus of claim 21, wherein at least a tip of thesample probe is made from a hydrophobic material.
 34. The flow cytometerapparatus of claim 33, wherein the hydrophobic material comprises one ofTeflon® (poly(tetrafluoroethylene) (PTFE)), Kynar® (polyvinylidenefluoride), Tefzel® (ethylene-tetrafluoroethylene copolymer),tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer resin (PFA), atetrafluoroethylene-hexafluoropropylene copolymer (EFP), or polyetherether ketone (PEEK).
 35. A flow cytometer apparatus, comprising: a flowcell having a first end and a second end; a first fluidic pathway havinga first end and a second end, wherein the second end of the firstfluidic pathway is coupled to the first end of the flow cell; a sampleprobe coupled to the first end of the first fluidic pathway; a firstpump in fluid communication with the first fluidic pathway; a secondfluidic pathway having a first end and a second end, wherein the firstend of the second fluidic pathway is coupled to the second end of theflow cell; a second pump in fluid communication with the second fluidicpathway; and a fault detection system configured to detect a clog in theflow cytometer apparatus, wherein the fault detection system comprisesnon-transitory computer-readable medium having stored thereoninstructions that, when executed by one or more processors, cause thefault detection system to perform functions comprising: detecting afirst plurality of events associated with a first aliquot from a firstsample well; and in response to a determination that a count of thefirst plurality of events is below a minimum count tolerance,determining that the flow cytometer apparatus is clogged.
 36. The flowcytometer apparatus of claim 33, wherein the first pump comprises asample pump, and wherein the second pump comprises a waste pump.
 37. Theflow cytometer apparatus of claim 35, further comprising: a thirdfluidic pathway coupled to the first end of the flow cell; and a thirdpump in fluid communication with the third fluidic pathway, wherein thethird pump comprises a sheath pump; and a sheath fluid reservoir coupledto the third fluidic pathway, wherein the sheath fluid reservoir isconfigured to hold a sheath fluid.
 38. A non-transitorycomputer-readable medium having stored thereon instructions that, whenexecuted by one or more processors of a flow cytometer apparatus, causethe flow cytometer apparatus to perform functions comprising: detectinga first plurality of events associated with a first aliquot from a firstsample well; determining a count of the first plurality of eventsassociated with the first aliquot; determining Whether the count of thefirst plurality of events is below a minimum count tolerance; if thecount of the first plurality of events is below the minimum counttolerance, then determining that the flow cytometer apparatus has aclog; and if the count of the first plurality of events is equal to orabove the minimum count tolerance, then detecting a second plurality ofevents associated with a second aliquot from a second sample well. 39.The non-transitory computer-readable medium of claim 38, wherein thefunctions further comprise: determining a count of the second pluralityof events determining whether the count of the second plurality ofevents is below a minimum count tolerance; if the count of the secondplurality of events is below the minimum count tolerance, thendetermining that the flow cytometer apparatus has a clog; and if thecount of the second plurality of events is equal to or above the minimumcount tolerance, then detecting a third plurality of events associatedwith a third aliquot from a third sample well of the flow cytometerapparatus.